Escherichia coli having accession No. PTA 1579 and its use to produce polyhydroxybutyrate

ABSTRACT

The present invention provides a novel genetically modified  Escherichia coli  JM109 bearing accession number PTA 1579, containing the gene coding for poly-beta-hydroxybutyrate synthesis and a method of using this bacterium to produce poly-beta-hydroxybutyrate to the extent of 60% or more of the cell weight.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. patent application Ser. No.09/772,304, now U.S. Pat. No. 6,756,222, filed Jan. 29, 2001, which isincorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to the field of recombinantdeoxyribonucleic acid (DNA) technology. Specifically, the inventionrelates to identification of the genes responsible forpoly-beta-hydroxybutyrate biosynthesis pathway from Streptomycesaureofaciens NRRL 2209, creation of a plasmid vector carrying the saidgene and expression of this gene in Escherichia coli designated as NCIM5128 and bearing ATCC Accession No. PTA 1579, which is used forsynthesis of polyhydroxybutyrate in recoverable amounts of at least 60%of dry bacterial cell mass.

BACKGROUND OF THE INVENTION

Lemoigne in 1926 discovered the presence of PHB(poly-beta-hydroxybutyrate) in Bacillus. This has been reported to bepresent in a multitude of other bacterial genera, including Azotobacter,Alcaligenes, Psuedonomas, Rhizobium, Chromatium, Acinetobacter,Rhodospirillum and some species of cyanobacteria. It is also reported tobe present in certain actinomycetes in very minute quantities. PHB issynthesized and stored by these microorganisms essentially as an energysource under stress conditions. PHB, a homopolymer ofD-(-)-3-hydroxybutyrate, has properties comparable to synthetic polymerslike polypropylene. PHB is commercially produced by fermentationtechnology using Alcaligenes eutrophus (Ralstonia eutropha) and ismarketed under the brand name Biopol. In the present global environmentawareness, as against synthetic polymers which are persistent by nature,PHB is bestowed with the property of biodegradability. Besides beingused in packaging industry, PHB has also been used as a source of chiralcenters for the organic synthesis of certain antibiotics, in drugdelivery and bone replacement applications. The biosynthesis of PHB hasbeen-studied extensively in Alcaligenes eutrophus, Rhodospirullumrubrum, Pseudomonas species and Azotobacter beijerinckii.β-ketothiolase, the first enzyme in the pathway and coded for by thephaA gene, first catalyzes the reversible condensation of two acetylcoenzyme A (CoA) molecules to acetoacetyl-CoA. Acetoacetyl-CoA is thenreduced to D-(-)-3-hydroxybutyryl-CoA by NADPH dependent acetoacetyl-CoAreductase which is coded for by phaB gene. D-(-)-3-hydroxybutyryl-CoAmonomer is then polymerized to PHB by PHB synthase coded for by the phaCgene. PHB in the bacterial cell accumulates as cytoplasmic inclusionswhen growth of the bacteria in culture is limited by a nutrient otherthan a carbon source. It may be oxygen deprivation, nitrogendeprivation, phosphate limitation, sulfate limitation and magnesiumlimitation. Once the limiting conditions are relaxed, PHB is metabolizeddown to preinduction levels. It has been shown that both β-ketothiolaseand acetoacetyl CoA reductase activities increase in response toPHB-stimulating limitation conditions.

Some of the U.S. Patents covering production and extraction of PHB frommicroorganisms include the following: U.S. Pat. No. 4,786,598 toLafferty et al. discloses a two-stage fermentation process where PHB isproduced using Alcaligenes latus, U.S. Pat. No. 4,705,604 to Vanlautemet al. discloses using 1,2 dichloroethane to simultaneously remove waterfrom the bacterial suspension by azeotropic distillation and extract PHBfrom the cells, U.S. Pat. No. 4,477,654 to Holmes et al discloseslimiting the nitrogen nutrient source to microbiologically accumulate3-hydroxybutyrate polymers, U.S. Pat. No. 4,433,053 discloses afermenting process for PHB accumulation using A. eutrophus where anutrient required for growth is limited, U.S. Pat. No. 4,336,334 toPowell et al. shows a microbiological process for producing PH usingMethylobacterium organophilum, U.S. Pat. No. 4,358,583 to Walker et al.discloses extracting PHB by first flocculating the cells by heat or pHtreatment then extracting with a suitable solvent, U.S. Pat. No.4,138,291 to Lafferty discloses bacterial strains assimilating variouscarbon sources and converting them to PHB, U.S. Pat. No. 5,518,907 toDennis discloses Cloning and expression in Escherichia coli of theAlcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosyntheticpathway, U.S. Pat. No. 5,798,235 to Peoples, et. al., gene encodingbacterial acetoacetyl Co-A reductase and U.S. Pat. No. 5,650,555 toSomerville et. al. discloses transgenic plants producingpolyhydroxyalkanoates. U.S. Pat. No. 5,512,456 to Dennis disclosesmethod for the improved production and recovery ofpoly-beta-hydroxybutyrate from transformed Escherichia coli, U.S. Pat.No. 5,250,430 discloses Polyhydroxyalkanoate polymerase from Zoogloearamigera.

Among the prokaryotes, the actinomycetes constitute an important part ofthe microbial community responsible for the degradation and recycling ofnatural substrates. Accumulation of PHB has been reported from ninedifferent strains of Streptomyces by Kannan and Rehacek (Formation ofpoly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126–129, 1970). A possible role in thew biosynthesis ofpolyketide-derived phenolic metabolites such as actinorhodin orantimycin has been suggested by Kannan and Rehacek (Formation ofpoly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126–129, 1970) and Packter and Flatman (Characterization ofacetoacetyl-CoA reductase (3-oxoreductase) from Streptomyces coelicolor,its possible role in polyhydroxybutyrate biosynthesis. Biochem. Soc.Trans., 11: 598–599, 1983). Streptomyces aureofaciens NRRL2209 is a verypoor accumulator of PHB and has been reported by Kannan and Rehacek(Formation of poly-beta-hydroxybutyrate by actinomycetes. Indian J.Biochem., 7: 126–129, 1970) to accumulate PHB up to 1.10% of the drycell mass. These bacteria are not as amenable as Escherichia coli togenetic manipulations and certainly are not as well characterized.Escherichia coli as a host cell has been exploited for producingmolecules like the human growth hormone, insulin and interferon.

Thus, although polyhydroxybutyrate and other polyalkanoate biosynthesisgenes have been isolated and characterized in various organisms such asRalstonia eutropha (formerly known as Alcaligenes entrophus), there areseveral disadvantages in the use of these genes for production ofpolyalkanaoates on a large scale. For instance, when these genes arecloned into bacteria such as E.coli, the eventual production ofalkanoate is not substantial. Further, there is no instance in the priorart wherein DNA fragments are isolated from actinomycetes and expressedin a heterogeneous host for production of polyhydroxybutyrate.

In order to make PHB production regulatable; a need exists for cloningof the PHB biosynthetic pathway from Streptomyces aureofaciens NRRL2209,its introduction and expression in Escherichia coli.

OBJECTS OF THE INVENTION

The main object of the invention is to provide a method for cloning ofgenes coding for poly-beta-hydroxybutyrate biosynthesis pathway fromStreptomyces aureofaciens NRRL2209 and expression thereof in Escherichiacoli.

Another object of the present invention is to identify the genomicsequences responsible for poly-beta-hydroxybutyrate biosynthesispathway, from Streptomyces aureofaciens NRRL 2209.

Yet another object of the present invention is to clone the genesresponsible for poly-beta-hydroxybutyrate biosynthesis pathway, in amulticopy plasmid vector, thus creating a new vector which carries thenucleotide sequence responsible for the PHB synthesis pathway.

Yet another object of the present invention is to transform Escherichiacoli with a multicopy plasmid vector for expression of the PHBbiosynthesis pathway genes in the bacterial host.

Another object of the present invention is to produce PHB usingtransformed Escherichia coli in recoverable quantities.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a method for isolation ofgenes coding for poly-beta-hydroxybutyrate (PHB) biosynthesis pathwayfrom Streptomyces aureofaciens NRRL2209, expressing the said genes inEscherichia coli and producing PHB using the transformed Escherichiacoli to the extent of 60% or more of the cell weight.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a method for the productionof poly-beta hydroxybutyrate (PHB) using recombinant Escherichia coli,said method comprising the steps of:

-   i) isolating the DNA sequence coding for the    poly-beta-hydroxybutyrate (PHB) biosynthetic pathway, from    Streptomyces aureofaciens NRRL2209,-   ii) cloning the DNA sequence coding for PHB pathway into a plasmid    vector pGEM-3Z to obtain a multicopy vector designated as pSa240,-   iii) transforming Escherichia coli JM109 with the plasmid vectyor    pSa240 to obtain recombinant Escherichia coli JM109 bearing    accession No.PTA1579 and harbouring the gene responsible for    production of PHB, and-   iv) culturing recombinant Escherichia coli JM109 in a conventional    medium containing glycerol and recovering poly-beta-hydroxybutyrate.

In an embodiment, the nucleic acid fragment coding forpoly-beta-hydroxybutyrate synthesis pathway is a 4.826 Kb long fragment(SEQ ID NO:1).

In another embodiment the nucleic acid fragment coding for PHB pathwayis isolated from Streptomyces aurefaciens NRRL2209.

In yet another embodiment, the DNA sequence coding for PHB pathway iscloned into a multicopy plasmid vector named pGEM-3Z.

In another embodiment, the plasmid vector harbouring the gene coding forPHB pathway is pSa240.

In yet another embodiment, Escherichia coli JM109 is transformed withthe multicopy plasmid vector pSa240 at a temperature in the range of14°–18° C. in the presence of T4 DNA ligase enzyme.

In another embodiment, the recombinant Escherichia coli JM109 isdeposited with the American Type Culture Collection, USA and hasreceived accession No.PTA 1579. The deposit is made in compliance withthe Budapest Treaty requirements.

In yet another embodiment, the transformed recombinant Escherichia coliJM109 when cultured in medium containing glycerol expresses the saidbiosynthetic pathway gene by producing poly-beta-hydroxybutyrate inrecoverable quantities of at least about 60% of the dry cell mass of theEscherichia coli JM109 bacterial host.

The Escherichia coli JM109 bacterial host has been deposited with ATCCand bears Accession Number PTA No.1579.

The transformed and genetically modified strain Escherichia coli JM109harbors the plasmid pSa240 expresses gene for ampicillin resistance andthe polyhydroxybutyrate biosynthetic operon obtained from Streptomycesaureofaciens NRRL 2209. The original Escherichia coli JM109 strain wasprocured from ATCC and bears accession number 53323.

As said earlier, polyhydroxybutyrate and other polyhydroxyalkanoatebiosynthesis genes have been isolated, characterized and patented fromorganism like Ralstonia eutropha (formerly known as Alcaligeneseutrophus), Rhodospirillum rubrum, Zoogloea ramigera. The Applicantshave now have isolated, cloned, sequenced and characterized a 4.826 kbSau3A I restriction endonuclease fragment from the genomic DNA ofStreptomyces aureofaciens NRRL 2209 (SEQ ID NO:1) and this fragmentcarries all the genetic information required for the synthesis ofpolyhydroxybutyrate upon it being used to transform Escherichia coliJM109. This is the first instance of its kind where DNA fragmentisolated from a member of actinomycetes has been expressed in aheterologous host for the production of polyhydroxybutyrate. Thetransformed Escherichia coli harboring the 4.826 kb Sau3A I restrictionendonuclease fragment (SEQ ID NO:1) preferentially uses glycerol as acarbon source for the synthesis of polyhydroxybutyrate. The putativepolyhydroxybutyrate biosynthesis genes that the applicants have isolateddoes not show significant sequence similarity at amino acid level withany of the reported polyhydroxybutyrate biosynthesis gene sequences.While Streptomyces aureofaciens NRRL 2209 accumulates only about 1%polyhydroxybutyrate, the DNA fragment that the applicants have isolatedfrom this organism and introduced into Escherichia coli JM109 supportsproduction and accumulation of polyhydroxybutyrate to the extent of 60%of dry cell mass of this heterologous host. The novelty of the patent isthe isolation, cloning, sequencing and heterologous statement of a 4.826kb DNA sequence from Streptomyces aureofaciens NRRL2209 (SEQ ID NO:1)for the production of polyhydroxybutyrate.

The novelty of the invention resides in isolation, cloning, sequencingand characterization of a 4.826 kb Sau3A I restriction fragment from thegenomic DNA of Streptomyces aureofaciens NRRL 2209 (SEQ ID NO:1). TheSau3A I DNA fragment (SEQ ID NO:1) harbors genes responsible for thesynthesis of polyhydroxybutyrate. This Sau3A I DNA fragment (SEQ IDNO:1) when cloned and introduced into Escherichia coli JM109 as plasmidvector pSa240 supports the synthesis of polyhydroxybutyrate to theextent of at least 60% dry mass of the bacterial cell. The recombinantEscherichia coli JM109 (ATCC PTA-1579) utilizes glycerol as a carbonsource for the synthesis of polyhydroxybutyrate.

However, with the use of other carbon sources individually or incombination it may be possible to order the synthesis of the other homo-or co-polymers of hydroxyalkanoates.

To describe in detail, the present invention relates to a novel methodfor improved production and recovery of PHB using Escherichia coliJM109. The invention is described in detail by the accompanying drawingsillustrated herein below and the following description and examples.

FIG. 1 represents colony hybridization of Streptomyces aureofaciensNRRL2209 Sau3A I subgenomic library with the 1.55 kilobase Nco I/Stu Irestriction fragment of the phaC gene from Ralstonia eutropha.

FIGS. 2 a and 2 b represent respectively the agarose gel electrophoresisof twenty four phaC positive clones and their Southern blot analysisusing the 570 base pair Sac I DNA fragment spanning the central regionof the phaC gene of Ralstonia eutropha as the probe.

FIG. 3 represents the Southern blot analysis of twelve phaC positiveclones hybridized to the 300 base pair Nsp(7524) V/Nco I restrictionfragment spanning the 5′ region of the phaC gene from Ralstoniaeutropha.

FIG. 4 represents the Southern blot analysis of the twelve phaC positiveclones hybridized to the 430 base pair Sac I/Stu I restriction fragmentspanning the 3′ region of the phaC gene from Ralstonia eutropha.

FIG. 5 represents the Southern blot analysis of the twelve phaA positiveclones hybridized to the 1.0 kilobase Stu I restriction fragmentspanning the phaA gene from Ralstonia eutropha.

FIG. 6 represents the fluorescence photomicrographs of Escherichia coliJM109 harboring pSa240 showing orange fluorescence due to presence ofintracellular PHB granules.

FIG. 7 represents the restriction endonuclease map of the pSa240 insert.

FIGS. 8A–B represents the nucleotide sequence of the 4.826 kilobaseSau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL2209(SEQ ID NO:1) cloned and present as an insert in the pSa240 plasmid.

FIG. 9 represents Southern blot analysis of genomic DNA fromStreptomyces aureofaciens NRRL2209 using the pSa240 insert as thehybridization probe.

FIG. 10 represent the gas chromatogram of PHB extracted from Escherichiacoli 109 harboring the pSa240 plasmid.

FIG. 11 represents nuclear magnetic resonance (NMR) spectrum of PHBisolated from Escherichia coli harboring pSa240 plasmid

FIG. 12 represents a graph showing time course of growth and PHBaccumulation by the recombinant Escherichia coli JM109 harboring thepSa240 plasmid and bearing accession number NCIM 5128 (ATCC PTA 1579).

In this invention, building a subgenomic library of Streptomycesaureofaciens NRRL2209 was commenced with the genomic DNA being partiallydigested with the restriction endonuclease Sau3A I, DNA fragments 2–6kilobase in size isolated, purified and shot gun cloned by ligation tothe BamH I restriction site of pGEM-3Z plasmid vector. The ligation mixwas later used to transform Escherichia coli JM109 and develop asubgenomic library. The subgenomic library was screened for the presenceof the phaC gene using the 1.55 kilobase Nco I/Stu I DNA fragmentspanning the phaC gene from Ralstonia eutropha as the probe and usingstandard colony hybridization procedures. Twenty four clones which gavestrong positive signal were isolated and the recombinant plasmid DNA waspurified from each of them.

In another embodiment of the present invention the insert from the eachof the recombinant plasmids was released by restriction digestion withEcoR I/Pst I. The samples were electrophoresed in an agarose gel andSouthern hybridization was done using standard procedures. The 570 basepair Sac I DNA fragment spanning the central region of the phaC gene ofRalstonia eutropha was used as the probe. On the basis of stronghybridization signal and large insert size twelve clones were selected.

In yet another embodiment of the present invention the above said twelvephaC positive clones were further characterized for the presence of thephaC gene by sequentially first hybridization with the 300 base pairNsp(7524) V/Nco I restriction fragment and later with the 430 base pairSac I/Stu I restriction fragment both from the phaC gene of Ralstoniaeutropha. The twelve phaC clones were further characterized for thepresence of the phaA gene by standard hybridization procedures using the1.0 kilobase Stu I DNA fragment spanning the phaA gene of Ralstoniaeutropha as the hybridization probe. Three phaA positive clones whichgave strong hybridization signal were selected.

In still another embodiment of the present invention the above threeclones which gave positive hybridization signals for the phaC and thephaA genes were analyzed for PHB production. One Escherichia coli JM109clone harboring the designated pSa240 plasmid was found to produce PHBin substantial and recoverable quantities, and is deposited with ATCC,U.S.A. and afforded accession number ATCC PTA 1579

In another embodiment of the present invention the insert in the pSa240plasmid was mapped with restriction enzymes and found to beapproximately 5.0 kilobase in size.

In yet another embodiment of the present invention the insert in thepSa240 plasmid was subjected to nucleotide sequence and found to be4.826 kilobase in size.

Experiments have been conducted which include the isolation, cloning andsequencing of the PHB biosynthetic pathway from Streptomycesaureofaciens NRRL2209, and the production and accumulation of PHB inrecombinant Escherichia coli to a high internal concentration. Allchemicals used in the experiments were obtained from the Sigma ChemicalCompany, United States Biochemicals, New England Biolabs and PromegaCorporation all from the U.S.A and Amersham, U.K. Streptomycesaureofaciens NRRL 2209 was from National Regional Research Institute,Perioa, Ill., U.S.A. and Escherichia coli JM109 was obtained from theAmerican Type Culture Collection (ATCC), Manassas, Va., U.S.A. LuriaBertani broth (LB) and antibiotics were prepared according to Sambrooket al (Molecular Cloning: a laboratory manual, 2^(nd) edition, ColdSpring Harbor Laboratory, New York, 1989). The pGEM-3Z plasmid vectorwas obtained from Promega Corporation, Madison, Wis., U.S.A.

A subgenomic library of Streptomyces aureofaciens NRRL2209 wasconstructed by inserting 2–6 kilobase Sau3A I digested genomic DNAfragments in pGEM-3Z plasmid vector, followed by transformation ofEscherichia coli JM109. Total Streptomyces aureofaciens NRRL2209 genomicDNA was prepared by the protoplast lysis method described by Tripathiand Rawal (Simple and efficient protocol for isolation of high molecularweight DNA from Streptomyces aureofaciens. Biotechnol. Tech., 12:629–632, 1998). Basic cloning techniques were followed as enumerated bySambrook et al (Molecular Cloning: a laboratory manual, 2^(nd) edition,Cold Spring Harbor Laboratory, New York, 1989). The genomic DNA waspartially digested with the restriction endonuclease Sau3A I. Therestriction digested DNA was electrophoresed in 1.0% agarose gel and DNAfragments in the 2–6 kilobase range were excised from the gel, andpurified by phenol extraction and ethanol precipitation. The plasmidpGEM-3Z DNA was linearized by restriction digestion with the restrictionendonuclease BamH I, and purified by phenol extraction and ethanolprecipitation. The Sau3A I partially digested genomic DNA fragments andthe BamH I digested plasmid were subjected to ligation overnight at 16°C. using T4 DNA ligase (Promega, U.S.A) as per manufacturersrecommendations The ligated DNA was used to transform Escherichia coliJM109. The bacteria were plated onto LB plates containing ampicillin.The resultant ampicillin resistant bacterial colonies were used as thesubgenomic library. The subgenomic library was screened by colonyhybridization using the 1.55 kilobase Nco I/Sac I DNA fragment fromRalstonia eutropha spanning the phaC gene (which codes forpolyhydroxybutyrate synthase enzyme) as the hybridization probe (FIG.1). The colony hybridization procedure was followed according toSambrook et al. (Molecular Cloning: a laboratory manual, 2^(nd) edition,Cold Spring Harbor Laboratory, New York, 1989). Among the phaC positivecolonies, twenty four positive clones were selected on the basis ofstrong hybridization signal.

Plasmid DNA was isolated from the twenty four phaC positive recombinantEscherichia coli JM109, restriction digested with EcoR I and Pst I torelease the insert. The restriction digested DNA samples wereelectrophoresed on an agarose gel (FIG. 2 a), blotted onto Hybond N⁺membrane (Amersham, U.K) and screened by Southern hybridization usingthe 570 base pair Sac I DNA fragment spanning the central region of thephaC gene from Ralstonia eutropha as the hybridization probe (FIG. 2 b).Standard procedures were used for probe preparation and Southernhybridization as described by Sambrook et al. (Molecular Cloning: alaboratory manual, 2^(nd) edition, Cold Spring Harbor Laboratory, NewYork, 1989). The probe was made radioactive by random primer extensionmethod using Megaprime DNA labeling kit from Amersham, U.K. Both theinsert and the vector gave hybridization signal. On the basis of stronghybridization signal and large size of the insert twelve clones werefurther selected for analysis.

In another experiment the plasmid DNA from the twelve selected cloneswas again restriction digested with EcoR I and Pst I, electrophoresed in1% agarose gel, blotted onto Hybond N⁺ membrane (Amersham, U.K) andsubjected to Southern hybridization using the 300 base pair Nsp (7524)V/Nco I restriction fragment from Ralstonia eutropha which spans the 5′region of the phaC gene as the hybridization probe (FIG. 3). The probewas radioactive labeled by random primer extension method usingMegaprime labeling kit of Amersham, U.K. No hybridization signal wasseen from the insert of any of the twelve clones, however, the probehybridized to the vector. The hybridization probe was stripped from theblot using standard methods and the blot was then rehybridized with the430 base pair Sac I/Stu I DNA fragment spanning the 3′ region of thephaC gene from Ralstonia eutropha. The probe was made radioactive byrandom primer extension method using Megaprime labeling kit of Amersham,U.K. While the inserts gave positive signal to the probe, the linearizedvector gave very faint signal (FIG. 4).

In yet another experiment the plasmid DNA from the twelve selectedclones was again restriction digested with EcoR I and Pst I,electrophoresed in 1% agarose gel, blotted onto Hybond N⁺ membrane(Amersham, U.K) and subjected to Southern hybridization using the 1.0kilobase Stu I DNA fragment spanning the phaA gene from Ralstoniaeutropha as the probe. This gene codes for the enzyme β-ketothiolase.The probe was made radioactive by random primer extension method usingMegaprime DNA labeling kit from Amersham, U.K. Inserts from all theclones gave varying degree of hybridization signals with this probe(FIG. 5). The linearized plasmid vector also gave hybridization signal.

Based on the size of the insert and the strength of the hybridizationsignal of the recombinant clones, three clones designated pSa005, pSa067and pSa240 were selected for further analysis. The individualrecombinant Escherichia coli JM109 harboring the above said plasmidswere grown in basal medium supplemented with ampicillin and 1% glycerol,stained with Nile blue A and observed for orange fluorescence at anexcitation wavelength of 460 nm. No fluorescence was observed either inuntransformed Escherichia coli JM109 or in recombinant Escherichia coliJM109 harboring pSa005 or pSa067 plasmid. Escherichia coli cellsharboring the pSa240 plasmid, however, gave intense orange fluorescence(FIG. 6).

The pSa240 plasmid DNA was isolated from the recombinant Escherichiacoli JM109 in large quantities using standard alkali lysis method(Sambrook et al. Molecular Cloning: a laboratory manual, 2^(nd) edition,Cold Spring Harbor Laboratory, New York, 1989). The insert cloned intothe BamH I site of the pGEM-3Z plasmid vector was mapped with differentrestriction enzymes to: locate various restriction endonuclease siteswithin it, determine the size of different restriction fragments and todetermine the total size of the insert in the pSa240 plasmid (FIG. 7).In this manner the total size of the insert DNA was found to beapproximately 5.0 kilobase.

The approximate 5.0 kilobase DNA insert from Streptomyces aureofaciensNRRL 2209 in the pSa240 plasmid was bidirectionally sequenced bytransposon insertion system. Transposons were randomly inserted usingtransposon insertion kit from Epicentre Technologies Corporation, U.S.A.DNA sequencing reactions were performed using ABI Prism BigDyeTerminator Cycle Sequencing Kit from PE ABI, Connecticut, U.S.A. Thesize of the insert was found to be 4826 base pairs (FIG. 8).

Southern blot analysis by the method of Sambrook et al. (MolecularCloning: a laboratory manual, 2^(nd) edition, Cold Spring HarborLaboratory, New York, 1989) was performed to demonstrate presence of thePHB biosynthetic pathway in Streptomyces aureofaciens NRRL2209. Thegenomic DNA from the microorganism was extracted and digested withrestriction endonucleases Apa I, Sal I, Sma I, BamH I and Nco I. The4.826 kilobase insert released from pSa240 plasmid by restrictiondigestion with EcoR I and Pst I was gel purified, radioactive labeledand used as a probe. The PHB biosynthetic pathway is located on anapproximate 20 kilobase DNA fragment flanked by BamH I and Nco Irestriction endonuclease sites (FIG. 9).

Twenty four hour cultures of Escherichia coli harboring the pSa240plasmid insert were centrifuged to collect the cell mass. Total cellmass was esterified in presence of n-propanol, and subjected to gaschromatography (FIG. 10) to establish the presence of PHB in therecombinant Escherichia coli cells.

PHB extracted from Escherichia coli harboring pSa240 plasmid wasdissolved in deuteriated chloroform and analyzed by NMR (FIG. 11) whichestablished the chemical identity of PHB. Experiments on PHB productionrevealed that Escherichia coli harboring the pSa240 plasmid cloneproduced intracellular PHB to substantial levels. These levelsapproached at least 60% of the bacterial cell dry weight in 48 hours ofgrowth in culture. The high levels of expression obtained implies hightranscriptional and/or translatinal activity in the recombinantEscherichia coli. PHB is produced by the Escherichia coli harboring thePHB pathway from Streptomyces aureofaciens NRRL2209 under non-inducedconditions. Basal medium broth inoculated with Escherichia coliharboring the pSa240 plasmid and grown in presence of 1% glycerol as thesole carbon source accumulate PHB. PHB accumulation is higher inpresence of glycerol rather than in presence of glucose, sucrose ormolasses. The Escherichia coli strain harboring the pSa240 plasmid whichcarries a 4.826 kilobase PHB biosynthetic pathway from Streptomycesaureofaciens NRRL2209 which was produced according to the techniquesdescribed above has been deposited with the American Type CultureCollection, Manassas, Va., U.S.A. on Mar. 28, 2000 and bears accessionnumber ATCC PTA 1579. The advantage of the smaller multicopy plasmidpSa240 is its ability to produce more copies per bacterial cell. Whilethe invention has been described in terms of cloning the PHBbiosynthetic pathway from Streptomyces aureofaciens NRRL2209 intoEscherichia coli those skilled in the art of recombinant DNA technologywill recognize that other microorganisms produce PHB and that the PHBbiosynthetic pathway can be cloned into Escherichia coli in a mannercontemplated within the spirit and scope of the appended claims.

The following examples are given by way of illustration and thereforeshould not be construed to limit the scope of the present invention.

EXAMPLE 1

The genomic DNA from Streptomyces aureofaciens NRRL2209 was incubated inpresence of restriction endonuclease Sau3A I at 37° C. for 3 hours. Thedigested DNA was electrophoresed on 1% agarose gel and DNA fragments inthe size range 2–6 kilobase were isolated and purified by process ofphenolization and precipitation with ethanol.

EXAMPLE 2

The pGEM-3Z plasmid vector was restriction digested with the restrictionendonuclease BamH I for 1 hour at 37° C. The linearized plasmid wasobtained.

EXAMPLE 3

The linearized pGEM-3Z plasmid (example 2) and the 2–6 kilobase Sau3A Irestriction fragments from Streptomyces aureofaciens NRRL2209(example 1) were ligated at 16° C. with the help of T4 DNA ligase for 16hours.

EXAMPLE 4

The ligation product (example 3) was used to transform Escherichia coliJM109 cells which were then plated onto LB plates containing ampicillin.Recombinant Escherichia coli JM109 colonies grew in presence of theantibiotic selection.

EXAMPLE 5

The recombinant Escherichia coli JM109 (example 4) colonies were blottedonto Hybond N⁺ membrane (Amersham, U.K.). The master plates were storedunder refrigeration.

EXAMPLE 6

The 1.55 kilobase Nco I/Sac I restriction fragment from Ralstoniaeutropha spanning the phaC gene was purified and radioactive labeledusing Megaprime DNA labeling kit (Amershamn, U.K.), denatured by heatingat 100° C. for 5 minutes and snap chilled on ice.

EXAMPLE 7

The Hybond N⁺ membrane (example 5) was placed with the colony side up ona pad of absorbent paper soaked in denaturing solution (1.5 M NaCl, 0.5M NaOH). After 7 minutes the blot was removed and placed in neutralizingbuffer (3 M NaCl, 0.5 M Tris-HCl, pH 7.4). After 3 minutes the blot waswashed in 2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0). The blotwas transferred to dry filter paper, air dried, baked at 80° C. The blotwas next prehybridized for 6–8 hours in hybridization buffer (1%crystalline BSA fraction V; 1.0 mM EDTA, pH 8.0: 0.5 M Na₂HPO₄, pH 7.2;7.0% SDS) at 60° C. in a hybridization incubator (Robin Scientific,U.S.A). The buffer was decanted off and fresh hybridization buffer wasadded along with the denatured radioactive labeled probe (example 6) andhybridization was carried out for 16–18 hours at 60° C. in ahybridization incubator (Robin Scientific, U.S.A). The buffer wasdecanted and multiple low stringency buffer (0.36 M NaCl; 0.02 M sodiumphosphate, pH 7.7; 2 mM EDTA, pH 8) washes were given to the membrane at60° C. for 15 minutes each. The membrane was then washed twice with highstringency buffer (36 mM NaCl; 2 mM sodium phosphate, pH 7.7; 0.2 mMEDTA, pH 8) at 62° C. for 15 minutes each. The membrane was next wrappedin cling wrap and exposed at −70° C. to X-ray film. The film was removedafter two days and developed for the visualization of phaC positivehybridization signals.

EXAMPLE 8

Twenty four phaC positive Escherichia coli JM109 colonies (identified asin example 7) were taken from the master plates (example 6) grown overnight in LB broth containing ampicillin and plasmid DNA was isolatedfrom each one (Sambrook et al. Molecular Cloning: a laboratory manual,2^(nd) edition, Cold Spring Harbor Laboratory, New York, 1989).

EXAMPLE 9

The 570 base pair Sac I restriction fragment from Ralstonia eutrophaspanning the central region of the phaC gene was purified andradioactive labeled using Megaprime DNA labeling kit (Amersham, U.K.),denatured by heating at 100° C. for 5 minutes and snap chilled on ice.

EXAMPLE 10

The plasmid DNA (example 8) was digested with restriction endonucleasesEcoR I and Pst I for one hour. The digested samples were electrophoresedin 1% agarose gel. After electrophoresis the gel was rinsed withdeionized water and placed in depurination solution (0.25 N HCl) for 20minutes. The gel was again rinsed with deionized water and then placedin denaturation solution (example 7) for 45 minutes. The gel was againrinsed with deionized water and placed in neutralization solution(example 7) for 15 minutes. This step was repeated once. The gel wasplaced on HybondN⁺ membrane (Amersham, U.K) and placed in a vacuumblotting unit (Pharmacia, Sweden). The transfer of DNA to the membranewas affected by application of vacuum and using 20×SSC buffer (3 M NaCl,0.3 M sodium citrate, pH 7.0) for one hour as the elutant. The membranewas next separated from the gel, washed once with 2×SSC (example 7) andbaked in an oven at 80° C. for 2 hours. The membrane blot wasprehybridized and then hybridized (example 7) to the radioactive labeled570 base pair Sac I fragment of the phaC gene from Ralstonia eutropha,(examle 9) and the blot was exposed to X-ray film at −70° C. The filmwas removed after two days and developed to visualize the positivehybridization signals.

EXAMPLE 11

The 300 base pair Nsp(7524) V/Nco I restriction fragment from Ralstoniaeutropha spanning the 5′ region of the phaC gene was purified andradioactive labeled using Megaprime DNA labeling kit (Amersham, U.K.),denatured by heating at 100° C. for 5 minutes and snap chilled on ice.

EXAMPLE 12

Out of the twenty four clones analyzed (example 10), twelve wereselected on the basis of high molecular weight of the insert DNA andhybridization signal. Plasmid DNA from these clones was digested withrestriction enzymes EcoR I and Pst I for one hour each. The restrictiondigested samples were electrophoresed, blotted onto HybondN⁺ membrane(Amersham, U.K) and hybridized (example 7) with the 300 base pairNsp(7524) V/Nco I probe (example 11) and the blot was exposed to X-rayfilm at −70° C. for two days.

EXAMPLE 13

To strip off the 300 base pair Nsp(7524) V/Nco I probe, the membraneblot (example 12) was boiled in deionized water and left to stand in itfor thirty minutes.

EXAMPLE 14

The 430 base pair Sac I/Stu I restriction fragment from Ralstoniaeutropha spanning the 3′ region of the phaC gene was purified andradioactive labeled using Megaprime DNA labeling kit (Amersham, U.K.),denatured by heating at 100° C. for 5 minutes and snap chilled on ice.

EXAMPLE 15

The membrane blot (example 13) was rehybridized (example 7) with the 430base pair Sac I/Stu I probe (example 14) and exposed to X-ray film at−70° C. for two days.

EXAMPLE 16

The 1.0 kilobase Stu I restriction fragment from Ralstonia eutrophaspanning the phaA gene was purified and radioactive labeled usingMegaprime DNA labeling kit (Amersham, U.K.), denatured by heating at100° C. for 5 minutes and snap chilled on ice.

EXAMPLE 17

The plasmid DNA from each of the twelve clones selected on the basis ofhigh molecular weight of the insert DNA and hybridization signal(example 12) was digested with restriction enzymes EcoR I and Pst I forone hour. The restriction digested samples were electrophoresed, blottedonto HybondN⁺ membrane (Amersham, U.K) and hybridized (example 7) withthe 1.0 kilo base Stu I probe (example 16) and exposed to X-ray film at−70° C. for two days.

EXAMPLE 18

Three clones which gave strong hybridization signals (examples 10, 15,16) were selected and the Escherichia coli JM109 harboring these plasmidclones were grown for 24 hours in basal medium supplemented withampicillin and 1% glycerol. About 100 μl of the cells were smeared on aglass slide, heat fixed and stained with 1% aqueous solution of NileBlue A at 55° C. for 10 minutes. The slides were washed with tap waterand then with 8% aqueous acetic acid. The smear was covered with a coverslip and observed under a fluorescence microscope at an excitationwavelength of 460 nm. Only one clone designated pSa240 gave brightorange fluorescence suggesting presence of PHB granules in the bacterialcells.

EXAMPLE 19

The Escherichia coli JM109 harboring pSa240 plasmid vector was grown inbasal medium broth with ampicillin and 1% glycerol (example 18), peltedby centrifugation at 4,000 rpm and resuspended in 5 ml of methanol. Thecells were again pelleted by centrifugation and vacuum dried. The driedcells were transferred to a tightly sealable glass tube foresterification. A 2 ml volume of 1,2-dichloroethane, 2 ml n-propanolcontaining hydrochloric acid (1 volume concentrated hydrochloric acid+4volumes n-propanol) and 200 μl of internal standard (2.0 g benzoic acidin 50 ml n-propanol) were mixed and incubated for 4 hours in a waterbath at 85° C. The mixture was cooled, transferred to centrifuge tubesand centrifuged at 12,000 rpm for 15 minutes. 0.2 μl of the supernatantwas injected into a gas chromatograph and PHB in the esterefied cellmass was detected by flame ionization detector.

EXAMPLE 20

The Escherichia coli JM109 harboring pSa240 plasmid vector was grown inLB broth with ampicillin and 1% glycerol (example 18), pelted bycentrifugation at 4,000 rpm and resuspended in 5 ml of methanol. Thecells were pelleted again by centrifugation and vacuum dried. The driedcells were dispersed in a 1:1 mixture of sodium hypochlorite (0.15%aqueous solution) and chloroform. The cell dispersion was kept on ashaker for 1.5 hours at 37° C. The dispersion was centrifuged at 10,000rpm for 10 minutes. The heavy chloroform phase containing solubilizedPHB was recovered and PHB precipitated by addition of five volumes ofmethanol. The precipitate was recovered by filtration and air dried.

EXAMPLE 21

The air dried PHB recovered from recombinant Escherichia coli JM109cells harboring the pSa240 plasmid (example 20) was dissolved indeuteriated chloroform and analyzed by nuclear magnetic resonance toestablish the chemical identity of PHB.

The Main Advantages of the Present Invention are:

-   1. The newly identified 4826 base pair Sau3A I restriction fragment    from the genomic DNA of Streptomyces aureofaciens NRRL2209 (SEQ ID    NO:1) which carries the genes for PHB synthesis is isolated and    cloned.-   2. The newly identified 4826 base pair Sau3A I restriction fragment    from the genomic DNA of Streptomyces aureofaciens NRRL2209 (SEQ ID    NO:1) is cloned in a multicopy plasmid vector to create a new    plasmid vector pSa240 which carries the nucleotide sequence of the    PHB biosynthesis genes.

3. Escherichia coli JM109 when transformed with the pSa240 plasmidvector produces polyhydroxybutyrate (PHB) in recoverable quantities ofat least 60% of cell dry mass utilizing glycerol as the carbon source.

1. A method for producing poly-beta-hydroxybutyrate, said methodcomprising: (i) isolating a nucleic acid encoding the proteinsresponsible for poly-beta-hydroxybutyrate synthesis from Streptomycesaureofaciens NRRL2209 comprising β-ketothiolase, acetoacetyl-CoAreductase, and PHB synthase, wherein the nucleic acid comprises SEQ IDNO:1, (ii) cloning said nucleic acid into a plasmid vector to obtain arecombinant vector, (iii) transforming Escherichia coli with saidrecombinant vector to obtain recombinant Escherichia coli whichexpresses said proteins responsible for poly-beta-hydroxybutyratesynthesis, (iv) culturing said recombinant Escherichia coli in aconventional medium comprising glycerol and one or more substrates and(v) recovering said poly-beta-hydroxybutyrate.
 2. The method accordingto claim 1 wherein the nucleic acid encoding the Droteins responsiblefor poly-beta-hydroxybutyrate synthesis is a 4.826 Kb fragment.
 3. Themethod according to claim 1 wherein the plasmid vector is a multicopyplasmid vector.
 4. The method according to claim 1 wherein therecombinant vector is pSa240.
 5. The method according to claim 4 whereinthe Escherichia coli is transformed at a temperature in the range of14–18C in the presence of T4 DNA ligase enzyme.
 6. The method accordingto claim 1 wherein poly-beta-hydroxybutyrate is produced in recoverablequantities of at least about 60% (w/w) of the recombinant E. coli drycell mass.
 7. The method according to claim 3, wherein the multicopyplasmid vector is pGEM-3Z.